Phosphaplatin complexes and methods for treatment of proliferative diseases using the phosphaplatin complexes

ABSTRACT

Stable monomeric phosphaplatins, namely, (pyrophosphato)platinum(II) or platinum(IV) complexes containing a cis-cyclohexanediamine ligand or enantiomerically enriched or enantiopure trans-cyclohexanediamine ligands, and synthesis of these complexes, are provided. Efficacies and toxicities of the phosphaplatin compounds are determined toward a variety of cancers, including sensitive and resistant ovarian cancers, head and neck, and colon cancers. Compositions comprising the platinum complexes, and methods for treatment of proliferative diseases or disorders by means of the complexes or the compositions comprising them are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/701,313, filed Nov. 30, 2012, which is the national stage ofInternational Application PCT/US2011/038948, filed Jun. 2, 2011, whichInternational Application claims the benefit of priority to U.S.Provisional Application Ser. No. 61/351,514, filed Jun. 4, 2010. Each ofthe foregoing applications is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present application relates generally to pyrophosphato platinumcomplexes; methods of synthesis of the provided complexes; compositionscomprising the provided complexes; and to methods of treatingproliferative diseases using the provided complexes, compositionscomprising the provided complexes, or combinations thereof.

BACKGROUND OF THE INVENTION

In the year 2008, over 12 million people worldwide were diagnosed withcancer and over 7 million people died from cancer. In fact, cancer isthe leading cause of death in the developed world and the second leadingcause of death in developing countries (second only to HIV/AIDS). Once acancer is diagnosed, the prognosis of the patient depends greatly onfactors such as whether the cancer was diagnosed at an early stage,whether the cancer has spread throughout the body, and whether thecancer is or has become resistant to known chemotherapeutic regimens.

The platinum-based anticancer drugs cisplatin, carboplatin, andoxaliplatin, are widely used for treating a variety of cancers such asovarian cancer, testicular cancer, small-cell lung cancer, andcolorectal cancer. These compounds may be used in combination with othertherapeutic regimens, including radiation therapy, to treat an expandedarray of cancers. Currently, over 600 clinical trials in adjuvanttherapeutic modes utilizing platinum compounds underscore the potentialof platinum compounds to effectively treat a wide variety of othercancers. For example, recent breakthrough research suggests that adiabetic drug, rosiglitazone, may be effectively used in combinationwith carboplatin to treat multiple forms of cancer. This has now added anew dimension to the ever-growing applications of platinum-basedanticancer drugs, because most adjuvant therapies have been limitedprimarily to combinations of cancer or radiation drugs with other cancerdrugs. Thus, there remains an ongoing need for new platinum-basedanticancer drugs, as well as new applications for platinum-basedanticancer drugs.

Conventional platinum chemotherapeutics such as cisplatin initiateapoptosis at the G2 phase of the cell cycle predominantly throughtranscription inhibition and through replication inhibition processes,especially at high doses. Covalent binding to DNA through the N7 sitesof guanine and adenine bases, both by intra-strand and inter-strandmodes, is believed to be the key molecular event in triggering a cascadeof cellular responses leading to apoptosis (programmed cell death).Numerous challenges have been identified in understanding the complexityof the cellular and molecular metallo-biochemistry of cisplatin and themolecular mechanisms of cytotoxicity. Briefly, it has been noted thatplatinated DNA is at the heart of the initiation of cytotoxicity. Theplatinum-bound DNA is sequestered by high mobility proteins (HMG) fromundergoing repairs by the nucleotide excision repair (NER) enzymes.Furthermore, these Platinum—DNA adducts are believed to activate the p53transcription factor, to induce histone phosphorylation, and to triggerchromatin condensation.

Although platinum-based chemotherapeutics are widely used to treatcancers, their applications in large numbers of patients have beenlimited because of severe side effects such as nephrotoxicity,neurotoxicity, ototoxicity, myelosuppression, and acquired resistance toplatinum-metal drugs. For example, a significant percentage of patientsbecomes resistant to cisplatin treatment. Although carboplatin reducessome toxicity over cisplatin, it does not alleviate the resistance.Currently, oxaliplatin is approved to treat colorectal cancer, but itsresistance is largely unexplored.

The art lacks an understanding at the molecular level of the developmentof resistance to conventional platinum-based chemotherapeutics and waysto overcome such resistance. Although the understanding is incomplete,it is believed that the ability to repair DNA damage by excising boundplatinum from DNA mostly contributes to the resistance mechanisms. Othermechanisms implicated in contributing to resistance include, reducedintracellular accumulations of cisplatin due to decreased uptake linkedwith the down-regulation of expression of the copper transport protein,CTR1; increased efflux due to overexpression of cMOAT, ATP7A, and ATP7B;impaired downregulation of pro-apoptotic genes and up-regulation ofanti-apoptotic genes. On the other hand, up-regulation of CTR1 proteinhas been linked with increased ototoxicity. Alternation of MAPKs anddeactivation of platinum by glutathione and other small molecules andproteins, especially metallothionine, have also been postulated ascontributing factors towards resistance to platinum drugs. In light ofthe aforementioned, there is a need for ways to overcome resistance toplatinum drugs, including development of drugs that are not susceptibleto DNA repair mechanisms.

In various embodiments, U.S. Pat. No. 7,700,649 (Bose) and U.S. Ser. No.12/722,189 (Bose) meet some of the needs in the art by disclosingsynthetic routes and cancer treatment methods involving a new class ofplatinum complexes, namely pyrophosphato complexes having platinum(II)or platinum(IV) metal centers. The disclosed compounds and methods arepart of a drug development strategy based on creating a class ofplatinum antitumor agents that do not covalently bind DNA, therebynullifying DNA-repair based resistance. This strategy is a paradigmshift from conventional platinum drug development approaches, in whichDNA binding is the central theme in developing more efficient platinumanticancer agents.

Among the pyrophosphato platinum complexes disclosed in U.S. Pat. No.7,700,649 (Bose) and U.S. Ser. No. 12/722,189 (Bose) are racemictrans-(±)-1,2-cyclohexanediamine(pyrophosphato) platinum(II) and racemictrans-(±)-1,2-cyclohexanediamine-trans-dihydroxo(pyrophosphato)platinum(IV). Although these racemic complexes have been foundefficacious for treatments of some cancers, a need still exists forimproved drug development approaches, including but not limited to,improving efficaciousness of pyrophosphato platinum therapeutics,reducing toxicities of such therapeutics, and inhibiting cancer cellgrowth by improved targeting of genes engaged in killing cancer cells.

SUMMARY OF THE INVENTION

In various embodiments, the present application fulfills the foregoingneeds by disclosing a drug development strategy based upon enantiopureand enantioenriched monomeric pyrophosphato platinum complexes. Thus,the present disclosure provides the most effective forms ofpyrophosphato platinum complexes, as well as identifying target genesengaged in killing cancer cells and inhibiting cancer cell growth. Theprovided complexes are stable, show enhanced cytotoxicity, and greatereffectiveness than conventional anticancer agents. This drug developmentstrategy is also a paradigm shift from conventional platinum drugdevelopment approaches, wherein DNA binding remains the central theme.

Among the various embodiments, the present application providesisolated, monomeric ((cis or trans)-1,2-cyclohexanediamine)(dihydrogenpyrophosphato)platinum (II) and ((cis ortrans)-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogenpyrophosphato)platinum (IV) complexes, wherein the complexes areenantiopure or comprise an enantiomeric excess of thecis-1,2-cyclohexanediamine-based complex or one of the twodistinguishable trans-1,2-cyclohexanediamine-based complexes. Thus, thepresent disclosure provides platinum (II) and platinum (IV) complexesselected from (i) ((1R,2R)-1,2-cyclohexanediamine)(dihydrogenpyrophosphato)platinum (II) (referred to herein as“(1R,2R)-pyrodach-2”); (ii) ((1S,2S)-1,2-cyclohexanediamine)(dihydrogenpyrophosphato)platinum (II) (referred to herein as“(1S,2S)-pyrodach-2”); (iii) ((1R,2S)-1,2-cyclohexanediamine)(dihydrogen pyrophosphato)platinum (II) or((1S,2R)-1,2-cyclohexanediamine) (dihydrogen pyrophosphato)platinum (II)(which are superimposable mirror-image compounds and are referred tocollectively herein as “cis-pyrodach-2”); (iv)((1R,2R)-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogenpyrophosphato)platinum (IV) (referred to herein as“(1R,2R)-pyrodach-4”); (v)((1S,2S)-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogenpyrophosphato)platinum (IV) (referred to herein as“(1S,2S)-pyrodach-4”); and (vi)((1R,2S)-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogenpyrophosphato)platinum (IV) or((1S,2R)-1,2-cyclohexanediamine)-trans-dihydroxo(dihydrogenpyrophosphato)platinum (IV) (which are superimposable mirror-imagecompounds and are referred to collectively herein as “cis-pyrodach-4”);as well as pharmaceutically acceptable salts or solvates of any of(i)-(vi). Referring to the amino groups on the 1,2-cyclohexanediamineligand, in the compounds (i), (ii), (iv), and (v), the (1R,2R) and(1S,2S) stereochemistries represent amino groups in transconfigurations, whereas in compounds (iii) and (vi) (1R,2S) and (1S,2R)stereochemistries represent amino groups in cis configurations.

The present disclosure additionally provides, in some embodiments,compositions comprising a therapeutically effective amount of one ormore of the provided complexes and at least one pharmaceuticallyacceptable ingredient such as a carrier, diluent, adjuvant, or vehicle.

In yet other embodiments, the present disclosure provides methods fortreating one or more proliferative diseases by administering to asubject in need thereof a therapeutically effective amount of acomposition comprising one or more of the provided complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

Though the specification concludes with claims particularly pointing outand distinctly claiming the invention, it is believed that the presentinvention will be better understood from the following description takenin conjunction with the accompanying drawings, in which:

FIG. 1 shows circular dichroism spectra of stereoisomers of (I)(1R,2R)-pyrodach-2, (II) (1S,2S)-pyrodach-2, (III) cis-pyrodach-2, (IV)(1R,2R)-pyrodach-4, (V) (1S,2S)-pyrodach-4, (VI) cis-pyrodach-4, andtrans-(±)-pyrodach-2;

FIG. 2 depicts activities of (1R,2R)-pyrodach-2, (1S,2S)-pyrodach-2, andtrans-(±)-pyrodach-2 as determined from clonogenic assays by exposinghuman ovarian cancer cells (A2780) to various concentrations ofcompounds for 24 hours;

FIG. 3 depicts activities of (1R,2R)-pyrodach-2, (1S,2S)-pyrodach-2, andtrans-(±)-pyrodach-2 as determined from clonogenic assays by exposingcisplatin-resistant human ovarian cancer cells (OVCAR-10) to variousconcentrations of compounds for 24 hours;

FIG. 4 is a plot of average tumor size over a period of six weeks duringadministration of phosphaplatins according to embodiments disclosedherein, on human ovarian cancer cells in mice;

FIG. 5 shows efficacies of (1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4,described in detail below, against cisplatin-resistant human ovariancancer cells (OVCAR-10);

FIG. 6 shows plots of tumor size over time, comparing a control(PBS/Bic) administered once every other day for three days (“qodx3”),carboplatin dosed at 60 mg/kg (administered qodx3) and(1R,2R)-pyrodach-4 dosed at 40 mg/kg [administered once every day forthree consecutive days (“qdx3”)]; and

FIG. 7 shows efficacies of (1R,2R)-pyrodach-2 [administered qodx3 andonce every day for four consecutive days (“qdx4”)] and(1R,2R)-pyrodach-4 (administered qdx4), described in detail below,against human head-and-neck cancer (UMSCC10b).

DETAILED DESCRIPTION OF THE INVENTION

Specific embodiments of the present disclosure will now be described.The invention may, however, be embodied in different forms and shouldnot be construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the specification and appended claims, the singular forms “a,”“an,” and “the” are intended to include the plural forms as well, unlessthe context clearly indicates otherwise.

It is noted that terms like “preferably,” “commonly,” and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

The term “substantially” is used herein to represent the inherent degreeof uncertainty that may be attributed to any quantitative comparison,value, measurement, or other representation. The term “substantially” isused herein also to represent the degree by which a quantitativerepresentation may vary from a stated reference without resulting in achange in the basic function of the subject matter at issue. As such, itis used to represent the inherent degree of uncertainty that may beattributed to any quantitative comparison, value, measurement, or otherrepresentation, referring to an arrangement of elements or featuresthat, while in theory would be expected to exhibit exact correspondenceor behavior, may in practice embody something slightly less than exact.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about,” whichis intended to mean up to ±10% of an indicated value. Additionally, thedisclosure of any ranges in the specification and claims are to beunderstood as including the range itself and also anything subsumedtherein, as well as endpoints. Unless otherwise indicated, the numericalproperties set forth in the specification and claims are approximationsthat may vary depending on the desired properties sought to be obtainedin embodiments of the present invention. Notwithstanding that numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

As used herein, the term “phosphaplatin” refers generally to platinumcomplexes coordinated with a single bidentate pyrophosphato ligand.Phosphaplatins according to embodiments described herein may have thefollowing general structures (A) and (B):

in which L¹ and L² represent neutral ligands (independently selectedfrom NH₃; substituted or unsubstituted aliphatic amines; and substitutedor unsubstituted aromatic amines), or a single bidentate neutral ligand(selected from substituted or unsubstituted aliphatic or aromaticdiamines) with end groups L¹ and L², coordinated to the platinum metalcenter; L³ and L⁴ are ligands (selected from hydroxide, acetic acid,butyric acid, and alpha-hydroxy acids, amines or charged speciesthereof) coordinated to the platinum metal center. The pyrophosphatoligand may be neutral (not shown) or charged (shown). When charged, thepyrophosphato ligand is present with counterions, represented by Z⁺.Examples of Z⁺ include, without limitation, hydrogen; alkali metals suchas sodium and potassium; and monovalent organic moieties. Preferably, Z⁺is a counterion that results in a pharmaceutically acceptable salt.Whether charged or neutral, the general structure of platinum(II)complexes represented by (A) is square-planar, and the general structureof platinum(IV) complexes represented by (B) is octahedral.

In general, phosphaplatins do not readily undergo hydrolysis, aresoluble in aqueous solution at neutral pH, and are stable in aqueoussolution at neutral pH. Furthermore, phosphaplatins show generalcytotoxicity in cancer cell lines, and are effective in cell lines thatare resistant to one or both cisplatin and carboplatin. Accordingly,phosphaplatins are effective, and in some cases more effective, ininducing cancer cell death as compared to known platinum cancer drugs,and exhibit desirable stability and solubility in solutions that aresuitable for administration to patients. As used herein in reference tothe phosphaplatins of the invention, “stable” refers to the resistanceof the complexes to hydrolysis when maintained in aqueous solution at apH in the range from 6-8 for a period of time from between 2 and sixdays.

Unlike cisplatin, carboplatin, and related platinum-based anti-canceragents, phosphaplatins do not covalently bind DNA. Resistance tocisplatin, carboplatin, and related platinum anti-cancer agents isbelieved to originate from the efficient repair of DNA damage by avariety of enzymes including nuclear excision repair enzymes. However,because phosphaplatins do not covalently bind DNA, resistance towardsphosphaplatins due to the DNA repair mechanism is unlikely. Data suggestthat phosphaplatins trigger overexpression of fas and fas-relatedtranscription factors, some proapoptotic genes such as Bak and Bax, andtumor suppression genes such as PUMA and PTEN. Moreover, phosphaplatinsdown-regulate BCL2, an antiapoptotic gene. Western Blot experiments thatdeal with protein expressions transcribed by these genes show theparallel trend. In addition, the cellular binding of phosphaplatins isless than cisplatin, yet phosphaplatins exhibit high cytotoxicity. Thus,the present invention provides effective platinum anticancer agents thathave a different molecular target than those in the art.

The term “enantiomeric excess” is used herein according to its commonlyunderstood definition. That is, for two enantiomers A and B that may bepresent in a mixture in molar amounts M_(A) and M_(B), respectively, theenantiomeric excess E of the enantiomer present in a higher molar amountin the mixture may be expressed by the relation %E=|(M_(A)−M_(B))/(M_(A)+M_(B))×100|, where E>0%. A “racemic mixture” ofthe enantiomers A and B, which may be designated by abbreviations suchas “rac” and/or “(±)” (or simply lack any reference to enantiomers) hasE=0% because M_(A)=M_(B). As a further illustration, a mixtureconsisting of A and B, in which M_(A)=60% and M_(B)=40%, has anenantiomeric excess of A equal to 20%. The same mixture may be regardedin the alternative as a mixture consisting of 80% racemic mixture of Aand B in combination with 20% enantiopure A, inasmuch as each moleculeof B (40% of the mixture) may be paired with a molecule of A in themixture (40% of the mixture) to leave unpaired an excess of molecules ofA (20% of the mixture).

As used herein, the term “enantiopure” with regard to a molecule havingtwo enantiomers, A and B, refers to a compound or composition containingsubstantially only one of the enantiomers A or B, but not both A and B.For an “enantiopure” complex, 97%≤E≤100%.

As used herein, the term “enantioenriched” refers, in its broadestsense, to a compound or composition containing a molecule having twoenantiomers, A and B, such that the compound or composition has anenantiomeric excess of one of the enantiomers, either A or B. Thus, an“enantioenriched mixture of A and B” may refer to a mixture with anenantiomeric excess of A or to a mixture with an enantiomeric excess ofB, wherein 0%<E≤100% for either A or B. As illustrative examples, theenantiomeric excess of either A or B may be greater than 0.01%, greaterthan 1%, greater than 10%, greater than 25%, greater than 50%, greaterthan 75%, greater than 90%, greater than 98%, greater than 99%, greaterthan 99.9%, or even equal to 100%.

In various embodiments, provided herein are stable, monomericphosphaplatin complexes (and compositions comprising a therapeuticallyeffective amount of one or more of said complexes). In some embodiments,said complexes and compositions may be used in methods of treatingcancers, including but not limited to, cancers resistant to treatment byone or more of cisplatin, carboplatin, and oxaliplatin.

Complexes

In the various embodiments, provided are phosphaplatin complexesselected from the group consisting of:

(i) enantiopure (1R,2R)-pyrodach-2 having formula (I);

(ii) enantiopure (1S,2S)-pyrodach-2 having formula (II);

(iii) enantioenriched pyrodach-2 having an enantiomeric excess of either(1R,2R)-pyrodach-2 having formula (I) or (1S,2S)-pyrodach-2 havingformula (II);

(iv) cis-pyrodach-2 having formula (III);

(v) enantiopure (1R,2R)-pyrodach-4 having formula (IV);

(vi) enantiopure (1S,2S)-pyrodach-4 having formula (V);

(vii) enantioenriched pyrodach-4 having an enantiomeric excess of either(1R,2R)-pyrodach-4 having formula (IV) or (1S,2S)-pyrodach-4 havingformula (V); and

(viii) cis-pyrodach-4 having formula (VI)

In the complexes according to formulas (I)-(VI), the shorthand notation“pyrodach” refers to a 1,2-cyclohexanediamine(pyrophosphato)platinumcomplex (where “pyro” refers to the bidentate pyrophosphato ligand and“dach” refers to the bidentate 1,2-cyclohexanediamine ligand (as namedaccording to IUPAC conventions), known also as 1,2-diaminocyclohexane.The notation (1R,2R), (1S,2S), or cis before the term “pyrodach” refersto the sterochemical configuration of the chiral centers at the1-position and the 2-position of the 1,2-cyclohexanediamine ligand. Thenumber (i.e., 2 or 4) following the notation “pyrodach” refers to theoxidation number of the platinum center. That is “pyrodach-2” refers toa platinum(II) complex, and “pyrodach-4” refers to a platinum(IV)complex.

The phosphaplatins of formulas (I)-(VI), with platinum coordinated topyrophosphate and 1,2-cyclohexanediamine ligands, can exist as fourstereoisomers due to the possible cis- and trans-geometry of the twoamino (—NH₂) groups at the chiral carbon centers 1 and 2 of the diamineligand. These stereoisomers exhibit the (1R,2R)-, (1S,2S)-, (1R,2S)-,and (1S,2R)-configurations. The trans-ligand,trans-1,2-cyclohexanediamine, affords two enantiomers, having the(1R,2R)- and (1S,2S)-configurations, respectively. The cis-isomer inprinciple encompasses the (1R,2S)- and (1S,2R)-enantiomers, but thesetwo cis-isomers are equivalent, superimposable mirror imagesindistinguishable from each other structurally and chemically. Thus, thetwo enantiomers of the cis-isomer will be referred to hereinafter simplyas the “cis-isomer” and are referred to with a single formula.

The enantioenriched pyrodach-2 mixture (iii) and the enantioenrichedpyrodach-4 mixture (vii) both are characterized by an enantiomericexcess greater than zero of either the (1R,2R)-enantiomer or the(1S,2S)-enantiomer. The enantiomeric excess may vary and in exampleembodiments may be greater than 0.01%, greater than 1%, greater than10%, greater than 25%, greater than 50%, greater than 75%, greater than90%, greater than 98%, greater than 99%, greater than 99.9%, or evenequal to 100%. In example embodiments, the enantiomeric excess is of the(1R,2R)-enantiomer, for example, an enantiomeric excess of(1R,2R)-enantiomer greater than 90%. In further example embodiments, theenantiomeric excess is of the (1S,2S)-enantiomer, for example, anenantiomeric excess of (1R,2R)-enantiomer greater than 90%. In stillfurther example embodiments, the enantioenriched pyrodach-2 mixture (i)and/or the enantioenriched pyrodach-4 mixture (ii) are enantiopure ineither the (1R,2R)-enantiomer or the (1S,2S)-enantiomer.

In comparative data presented herein between the enantiopure complexesaccording to formulas (I), (II), (IV), and (V) and corresponding racemicmixtures disclosed in U.S. Pat. No. 7,700,649, hereinafter, a racemicmixture of (1R,2R)-pyrodach-2 and (1S,2S)-pyrodach-2 will be referred toby the shorthand notation “trans-(±)-pyrodach-2.” Likewise, a racemicmixture of (1R,2R)-pyrodach-4 and (1S,2S)-pyrodach-4 will be referred toas “trans-(±)-pyrodach-4.”

As a non-limiting example, the compounds of formulas (I)-(VI) may besynthesized from a starting material such ascis-(1,2-cyclohexanediamine) dichloroplatinum(II), which may be preparedby converting K₂PtCl₄ to K₂PtI₄ by the addition of potassium iodide. TheK₂PtI₄ may then be reacted with a 1,2-cyclohexanediamine having adesired stereochemistry, such as cis-1,2-cyclohexanediamine,trans-(1R,2R)-1,2-cyclohexanediamine,trans-(1S,2S)-1,2-cyclohexanediamine, or mixtures thereof. The resulting(1,2-cyclohexanediamine)diiodoplatinum(II) complexes then may betransformed to the corresponding(1,2-cyclohexanediamine)diaquaplatinum(II) complexes in situ by addingtwo equivalents of silver nitrate. The diaqua species[Pt(1,2-cyclohexanediamine)(H₂O)₂] then may be converted to thecis-dichloro [Pt(1,2-cyclohexanediamine)Cl₂] complexes by addition ofpotassium chloride.

It will be understood that other suitable reaction conditions may beused. In non-limiting examples, the starting1,2-cyclohexanediamine-platinum(II) complexes were reacted with excesspyrophosphate and the temperature may be from about 35° C. to about 45°C., or any preferred narrower range between 35° C. and 45° C. Goodresults have been obtained at 40° C. In some examples, the reaction maybe allowed to proceed from about 13 hours to about 16 hours, or anypreferred narrower range between 13 hours and 16 hours. Good resultshave been obtained at reaction times of 15 hours. In some examples, thepH can be from about 6 to about 7, from about 7 to about 8, and fromabout 8 to about 9. Good results have been obtained at pH of about 8.

The aqueous reaction mixture may be concentrated such that precipitatesof pyrophosphate do not form. It will be understood that the aqueousreaction mixture may be concentrated in any suitable manner. Forexample, the aqueous reaction mixture may be concentrated by rotaryevaporation.

Thereupon, the pH of the reaction mixture may be lowered rapidly to a pHof less than 2 by addition of a suitable acid. In some examples, nitricacid may be used to lower the pH. In some embodiments, the pH is in therange between about 1 to about 2. Good results have been obtained at pHof 1.

In some examples, the reaction mixture may be cooled to a temperature ofbetween 5° C. and room temperature (25° C.±2° C.) after concentratingthe reaction mixture. In other examples, the method also includescooling the reaction mixture to a temperature of between 5° C. and roomtemperature after lowering the pH of the reaction mixture.

To prepare the platinum(IV) complexes according to formulas (II) and(V), additional steps are required to attach the hydroxo ligands. Thus,in addition to the steps described above, to the reaction mixture may beadded hydrogen peroxide, and optionally a reagent selected from thegroup acetate salts, butyrate salts, and salts of alpha-hydroxy acids,after maintaining the reaction mixture at a temperature of about 30° C.to about 60° C. for a period of about 12 hours to about 18 hours at a pHfrom about 7 to about 9. The optional reagent that may be added togetherwith hydrogen peroxide prior to concentration of the reaction mixturemay be selected from sodium acetate, sodium butyrate, amines, and sodiumsalts of alpha-hydroxy acids. In other examples, the optional reagentadded together with hydrogen peroxide prior to concentration of thereaction mixture may be selected from potassium acetate, potassiumbutyrate, any monodentate amines such as ammonia, isopropyl amine, andothers, and potassium salts of alpha-hydroxy acids.

Compositions

In some of the various embodiments, additionally provided arecompositions comprising one or more of (a) a provided phosphaplatincomplex; (b) a pharmaceutically acceptable salt of (a); and (c) apharmaceutically acceptable solvate of (a). Said compositions mayadditionally comprise at least one pharmaceutically acceptableingredient selected from carriers, diluents, adjuvants, and vehicles,which generally refer to inert, non-toxic, solid or liquid fillers,diluents, or encapsulating materials unreactive with the phosphaplatins.These types of additives are well known in the art and are furtherdescribed below with regard to treatment methods. According to thevarious embodiments, a provided composition comprises one or more of:

(i) enantiopure (1R,2R)-pyrodach-2 having formula (I), orpharmaceutically acceptable salt or solvate thereof;

(ii) enantiopure (1S,2S)-pyrodach-2 having formula (II), orpharmaceutically acceptable salt or solvate thereof;

(iii) enantioenriched pyrodach-2 having an enantiomeric excess of either(1R,2R)-pyrodach-2 having formula (I) or (1S,2S)-pyrodach-2 havingformula (II), or pharmaceutically acceptable salts or solvates thereof;

(iv) cis-pyrodach-2 having formula (III), or pharmaceutically acceptablesalt or solvate thereof;

(v) enantiopure (1R,2R)-pyrodach-4 having formula (IV), orpharmaceutically acceptable salt or solvate thereof;

(vi) enantiopure (1S,2S)-pyrodach-4 having formula (V), orpharmaceutically acceptable salt or solvate thereof;

(vii) enantioenriched pyrodach-4 having an enantiomeric excess of either(1R,2R)-pyrodach-4 having formula (IV) or (1S,2S)-pyrodach-4 havingformula (V), or pharmaceutically acceptable salts or solvates thereof;

and(viii) cis-pyrodach-4 having formula (VI), or pharmaceuticallyacceptable salt or solvate thereof;

In some embodiments, a provided composition comprises one complex (orpharmaceutically acceptable salt or solvate thereof) having a formulaaccording to any of formulas (I)-(VI). In some embodiments, a providedcomposition is a multicomplex mixture of at least two complexes (orpharmaceutically acceptable salts or solvates thereof) having formulasaccording to any of formulas (I)-(VI). The multicomplex mixture maycomprise one, two, three, four, five, or six of the compounds accordingto formulas (I)-(VI), provided the multicomplex mixture is not a pureracemic mixture of (1R,2R)-pyrodach-2 and (1S,2S)-pyrodach-2 or a pureracemic mixture of (1R,2R)-pyrodach-4 and (1S,2S)-pyrodach-4.

Contemplated Methods

In still further embodiments, the complexes, compositions, or both,described above may be used alone, or with other pharmaceuticallyacceptable ingredients, in methods of treating proliferative diseases ordisorders (collectively, “diseases”). The provided methods compriseadministering to a subject in need thereof a therapeutically effectiveamount of a complex or composition described above. The subject may bean animal such as, for example, a mammal, including a human.Proliferative diseases contemplated to be treatable in humans includeovarian cancer, testicular cancer, small-cell lung cancer,non-small-cell lung cancer and head-and-neck cancers, skin cancer,pancreatic cancer, breast cancer, colon cancer, glioblastoma cancer. Insome embodiments, it is contemplated that the complexes and/orcompositions may be used in combination therapies involving concurrentor sequential treatment with known platinum-metal drugs such ascisplatin, carboplatin, and/or oxaliplatin. It is further contemplatedthat the complexes and/or compositions may be used to treat cancersresistant to treatment by one or more of cisplatin, carboplatin,oxaliplatin and/or used in combination with other treatment classes,including anti-mitotics such as taxanes, nucleoside analogs such asGemcitabine, anthracycline antibiotics such as Doxorubicin, or targetedtherapies such as monoclonal antibodies.

As described herein, the complexes of formulas (I)-(VI) have been shownto be as effective as, or more effective than, cisplatin andcarboplatin, thus providing a method of cancer treatment for patientswho previously lacked effective alternatives to cisplatin andcarboplatin treatment. However, a patient need not have previously beentreated with cisplatin or carboplatin to be treated with the providedcomplexes, compositions, and methods described herein. Administration ofthe treatment can be performed in a hospital or other medical facilityby medical personnel.

The complexes of formulas (I)-(VI) may be administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “therapeutic effective amount” for purposes herein isthus determined by such considerations as are known in the art. Theamount must be effective to achieve improvement including, but notlimited to, improved survival rate or more rapid recovery, orimprovement or elimination of symptoms and other indicators as areselected as appropriate measures by those skilled in the art. It iscontemplated that the complexes of the present invention may beadministered to animals, including mammals and humans alone or ascompositions. Moreover, it is contemplated that the complexes offormulas (I)-(VI) may be administered over an especially widetherapeutic window. As one illustrative example, it is contemplated thatone or more provided complexes may be administered in one or more dosesof from 5 mg/kg to 50 mg/kg; alternatively from 10 mg/kg to 50 mg/kg;alternatively from 20 mg/kg to 50 mg/kg; alternatively from 30 mg/kg to50 mg/kg; alternatively from 40 mg/kg to 50 mg/kg; alternatively from 45mg/kg to 50 mg/kg. Of course, one of skill in the art will appreciatethat therapeutic dosages may vary by complex administered, compositionadministered, and subject receiving the administered complex orcomposition. Thus, therapeutic doses greater than 50 mg/kg are alsocontemplated, as are therapeutic doses less than 5 mg/kg. The doses canbe single doses or multiple doses over a period of several days. As anillustrative example, it is contemplated that the complexes of formulas(I)-(VI) may be administered in one, two, three, four, five, six, ormore doses in one more days. It is also contemplated that the complexesmay be administered continuously over one or more days, such as by apump or drip. As another illustrative example, it is contemplated thatthe complexes may be administered for one, two, three, four, five, six,seven, eight, nine, ten, or more days.

In a method of treatment, the complexes of formulas (I)-(VI) can beadministered in various ways. It should be noted that they can beadministered as the complex and can be administered alone in aqueoussolution taking advantage of the excellent solubility of thesecomplexes, or as an active ingredient in combination withpharmaceutically acceptable carriers, diluents, adjuvants and vehicles.It is contemplated that the complexes can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, intratonsillar, and intranasaladministration as well as intrathecal and infusion techniques. Implantsof the complexes may also be useful.

When the complexes of formulas (I)-(VI) are administered parenterally,they generally will be formulated in a unit dosage injectable form(e.g., solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for the compositions.Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. However, anyvehicle, diluent, or additive used would have to be compatible with thephosphaplatin complexes.

Sterile injectable solutions can be prepared by incorporating thephosphaplatin complexes in the required amount of the appropriatesolvent with one or more of the other ingredients, as desired.

A pharmacological formulation comprising the phosphaplatins can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the phosphaplatin complexes can be administeredparenterally to the patient in the form of slow-release subcutaneousimplants or targeted delivery systems such as monoclonal antibodies,vectored delivery, iontophoretic, polymer matrices, liposomes, andmicrospheres. Many other such implants, delivery systems, and modulesare well known to those skilled in the art.

EXAMPLES

The described embodiments will be better understood by reference to thefollowing examples, which are offered by way of illustration and whichone skilled in the art will recognize are not meant to be limiting.

Example 1 Synthesis of (1R,2R)-pyrodach-2 [formula (I)]

As a starting material for forming a platinum(II) complex, cis-diiodo-or cis-dichloro-(trans-(1R,2R)-(−)-1,2-cyclohexanediamine)platinum(II)was formed by reacting K₂PtI₄ or, more preferably K₂PtCl₄, respectivelywith (1R,2R)-(−)-1,2-cyclohexanediamine. Thecis-diiodo-((1R,2R)-(−)-1,2-cyclohexanediamine)platinum(II) or,preferably, thecis-dichloro-((1R,2R)-(−)-1,2-cyclohexanediamine)platinum(II) then wasdissolved with sodium pyrophosphate decahydrate in distilled water, pH8, and the resultant mixture is incubated at 40° C. for 15 hours.Following the incubation period, the solution was concentrated by rotaryevaporation and was filtered to remove any unreacted starting material.Rapidly lowering the pH to approximately 1.0 by addition of 1-N nitricacid precipitated the product. The precipitation was completed bycooling at about 0° C., and the product was isolated by vacuumfiltration and washed with cold water and acetone. The synthesis yieldedenantiopure (1R,2R)-pyrodach-2.

Example 2 Synthesis of (1S,2S)-pyrodach-2 [formula (II)]

Enantiopure (1S,2S)-1,2-cyclohexanediamine(pyrophosphato) platinum(II)was prepared in a manner analogous to the method described in SynthesisExample 1, except that cis-diiodo- orcis-dichloro-(trans-(1S,2S)-(+)-1,2-cyclohexanediamine) platinum(II) wasused as a starting material in the place of cis-diiodo- orcis-dichloro-(trans-(1R,2R)-(−)-1,2-cyclohexanediamine) platinum(II),respectively. The synthesis yielded enantiopure (1S,2S)-pyrodach-2.

Example 3 Synthesis of (1R,2R)-pyrodach-4 [formula (IV)]

The starting material from Synthesis Example 1, i.e., cis-diiodo- orcis-dichloro-(trans-(1R,2R)-(−)-1,2-cyclohexanediamine) platinum(II) andsodium pyrophosphate decahydrate was dissolved in distilled water, pH 8,and the resultant mixture was incubated at 40° C. for 15 hours.Following the incubation period, an aliquot of 30% H₂O₂ was added to thereaction mixture, and the reaction mixture was allowed to react for anadditional 3 hours. The solution then was concentrated by rotaryevaporation and was filtered to remove any unreacted starting material.Rapidly lowering the pH to approximately 1.0 by addition of 1-N nitricacid precipitated the product. Precipitation was completed by cooling atabout 0° C., and the product was isolated by vacuum filtration andwashed with cold water and acetone. The synthesis yielded enantiopure(1R,2R)-pyrodach-4.

Example 4 Synthesis of (1S,2S)-pyrodach-4 [formula (V)]

Enantiopure(1S,2S)-1,2-cyclohexanediamine-trans-dihydroxo(pyrophosphato)platinum(IV) was prepared in a manner analogous to the method describedin Synthesis Example 3, except that the starting material from SynthesisExample 2, i.e., cis-diiodo- orcis-dichloro-(trans-(1S,2S)-(+)-1,2-cyclohexanediamine) platinum(II) wasused as a starting material in the place of cis-diiodo- orcis-dichloro-(trans-(1R,2R)-(−)-1,2-cyclohexanediamine) platinum(II),respectively. The synthesis yielded enantiopure (1S,2S)-pyrodach-4.

Example 5 Synthesis of cis-pyrodach-2 [formula (III)]

In a 500-mL round-bottom flask provided with a stirring bar, sodiumpyrophosphate decahydrate (0.400 g) was dissolved in distilled water(250 mL). The pH of the solution then was adjusted to 8.0 using 2-Mnitric acid. Then the solution was placed in a water bath at 40° C. andstirred with the magnetic bar. Then to the stirring solution,cis-dichloro-(cis-1,2-cyclohexanediamine)platinum(II) (0.100 g, 0.26mmol) was added. The mixture was allowed to react for 15 hours, and thenthe solvent was evaporated at 48° C. under vacuum to a volume of 5 mL.Then the mixture was passed through filter paper and the solution wascollected in a 10-mL vial provided with a stirring bar. The vial wasplaced in an ice bath over a stirring plate and with gentle stirring thepH was adjusted from an initial 6.5 to 2.0 using 2-N nitric acid. Oncethe lower pH was reached, a precipitate slowly developed. The stirringwas continued for an additional 5 minutes, after which the suspensionwas filtered through a medium-porosity fritted-glass filter that hasbeen kept in ice before its use. Then the solid was washed with coldwater (2 portions of 5 mL) and cold acetone (2 portions of 5 mL), andthe filter was left in a desiccator overnight. This produced a lightyellow powder (0.077 g, 0.16 mmol, 60% yield).

Example 6 Synthesis of cis-pyrodach-4 [formula (VI)]

In a 500-mL round-bottom flask provided with a stirring bar, sodiumpyrophosphate decahydrate (0.400 g) was dissolved in distilled water(250 mL). The pH of the solution was then adjusted to 8.0 using 2 Mnitric acid. Then the solution was placed in a water bath at 40° C. andwas stirred with the magnetic bar. To the stirring solutioncis-dichloro-(cis-1,2-cyclohexanediamine) platinum(II) (0.100 g, 0.26mmol) was added. The mixture was allowed to react for 15 hours, 3 mL of30% (w/w) H₂O₂ were added, and three additional hours of reaction timewere given. Then the solvent was evaporated at 48° C. under vacuum to avolume of 5 mL. Then the mixture was passed through filter paper, andthe solution was collected in a 10-mL vial provided with a stirring bar.The vial was placed in an ice bath over a stirring plate and was gentlystirred while the pH was adjusted from an initial 6.5 to 2.5 using 2-Nnitric acid. Soon after the lower pH was reached, a precipitate slowlydeveloped. The stirring was continued for an additional 5 minutes, andthen the suspension was filtered through a medium-porosity fritted-glassfilter that had been kept in ice before its use. The solid was washedwith cold water (2 portions of 5 mL) and cold acetone (2 portions of 5mL) and the filter was left in a desiccator overnight. This produced awhite powder (0.120 g, 0.23 mmol, 88% yield).

Example 7 Characterizations of the Phosphaplatins

All phosphaplatins synthesized according to the above Synthesis Examplesexhibit solubility greater than 40 mM/L in aqueous solution at neutralpH in PBS and bicarbonate buffer. (1R,2R)-pyrodach-2 and(1R,2R)-pyrodach-4, in particular, show remarkable stability at neutralpH in aqueous solution. Typically, no decomposition is observed withinseven days after dissolving the phosphaplatin compounds in water andobserving by ³¹P-NMR spectroscopy.

Compounds prepared according to the Synthesis Examples above werecharacterized by circular dichroism (CD) spectroscopy to verify isomericconfiguration, and by ³¹P-NMR and Mass Spectrometry to verify thecomposition. An additional CD spectra was run on a trans-(±)-pyrodach-2racemic mixture prepared according to the methods described in U.S. Pat.No. 7,700,649.

The CD spectra were recorded in phosphate buffer (50 mM) at pH 6.8. The(1R,2R)- and the (1S,2S)-forms of both pyrodach-2 and pyrodach-4 showedoptical activities attributable chirality, but the racemic mixture(trans-(±)-pyrodach-2) and the cis-isomers of both pyrodach-2 andpyrodach-4 did not exhibit any CD peaks.

The CD spectra are depicted in FIG. 1, in which the numbers inparentheses refer to the compound of the formula corresponding to thenumber in the parentheses. The concentrations of the compounds asanalyzed were: (I), 2.7 mM; (II), 2.5 mM; (III), 1.3 mM; (IV), 3.5 mM;(V), 2.9 mM; (VI), 2.7 mM; and trans-(±)-pyrodach-2, 3.5 mM.

Example 8 In Vitro Efficacy and Cell Survival Assay (Clonogenic Assay)

To determine the relative activities of the phosphaplatins of formulas(I)-(VI), each stereoisomer was tested through in vitro clonogenicassays using human ovarian cancer cells, human colon cancer cells, andhuman head-and-neck cancer cells. Human ovarian cancer cells, A2780 andA2780/C30 (cross resistant to 30 μM cisplatin and 100 μM carboplatin),were obtained from Dr. Thomas Hamilton (Fox Chase Cancer Center,Philadelphia, Pa.). Cells were cultured on monolayer using RPMI 1640supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.25 units/mLinsulin and penicillin/streptomycin (100 units/mL) in a 37° C. incubatorcontinuously gassed with 5% CO₂. Cells were subcultured using 0.0625%trypsin in HBSS to maintain cells in exponential cell growth.

Half-maximal inhibitory concentration (IC₅₀) values were determinedusing a clonogenic assay or a CyQUANT® cell proliferation assay. In theclonogenic assay, for example, 500-700 A2780 cells from a single cellsuspension were plated onto 60 mm petri plates 24 hours before treatmentwith the platinum compounds to permit cell attachment. On the day oftreatment with the platinum compounds described in the above SynthesisExamples, the medium was decanted and was replaced with the appropriateconcentration of phosphaplatin compounds (from 50 nM to 75 μM) at threedifferent time points, and the treated cells were placed back into the37° C. incubator for 24 hours. Triplicate plates were set up for eachplatinum compound concentration. After the 24-hour treatment, the mediumcontaining the platinum compounds was decanted and was replaced withfresh medium. These plates were returned to the 37° C. incubator for 7days for colony formation.

In the CyQUANT cell proliferation assay, the IC₅₀ values were determinedby measuring the DNA content using a CyQUANT® Cell Proliferation AssayKit (Invitrogen), which contains a green-fluorescent dye that exhibitsstrong fluorescence intensity when bound to cellular DNA. In theseexperiments, desired number of cells were exposed to phosphaplatins ofdifferent concentrations for 72 hr. before measuring the DNA content.Because the DNA content is proportional to the number of survivingcells, the assay provides a quantitative measure of proliferating cells.The technique is described in detail in Jones et al., “Sensitivedetermination of cell number using the CyQUANT cell proliferationassay,” J. Immunol. Methods, vol. 254, pp. 85-98 (2001).

The IC₅₀ data from clonogenic assays and/or CyQUANT cell proliferationassays are summarized in TABLES 1-5. In TABLES 1-5, except where actualerror values are given, each reported value is assumed to have an errornot greater than ±15% of the reported value; and unless otherwisespecified, the data were obtained from clonogenic assays.

TABLE 1 IC₅₀ values for phosphaplatin compounds on human ovarian cancercell lines: A2780, epithelial human ovarian cancer; and A2780/C30,epithelial human ovarian cancer resistant to 30 μM cisplatin and 100 μMcarboplatin IC₅₀ (μM) for various cell lines at various treatment timesA2780 A2780/C30 Compound 1 hour 24 hours 7 days 1 hour 24 hours 7 days(1R,2R)-pyrodach-2 (I) 1.0 ± 0.1 0.5 ± 0.1 6.3 1.1 ± 0.1(1S,2S)-pyrodach-2 (II) 1.1 ± 0.1 trans-(±)-pyrodach-2 22 ± 4 2.4 ± 0.248 ± 5 (comparative) (1R,2R)-pyrodach-4 (IV) 45 ± 5 13   4.9 11 ± 2 11.7(1S,2S)-pyrodach-4 (V) 5.2 trans-(±)-pyrodach-4 170 ± 20 155 ± 20(comparative) cis-pyrodach-2 (III)  0.3 ± 0.05 cis-pyrodach-4 (VI) 3.8cisplatin  7  100 (comparative) carboplatin 90 >200 (comparative)

TABLE 2 IC₅₀ values for phosphaplatin compounds on human ovarian cancercell lines: OVCAR-10, human ovarian cancer resistant to cisplatintreatment; and OVCAR-5, advanced human ovarian cancer cells IC₅₀ (μM)for various Human Ovarian Cancer Cell Lines at various treatment timesOVCAR-10 OVCAR-5 24 48 24 48 Compound hours hours hours hours(1R,2R)-pyrodach-2 (I) 0.42 15.4 (1S,2S)-pyrodach-2 (II) 6.9 4.5trans-(±)-pyrodach-2 4.6 12.2 (comparative) (1R,2R)-pyrodach-4 (IV) 10.219.8 (1S,2S)-pyrodach-4 (V) 5.6 trans-(±)-pyrodach-4 14.4 (comparative)cisplatin (comparative) 4.1 carboplatin (comparative) 26.7

TABLE 3 IC₅₀ values for phosphaplatin compounds on human head-and- neckcancer cell lines: USMCC10b, human head-and-neck cancer cell line; andUMSCC-10b/15s, cisplatin-resistant human head-and-neck cancer cell lineIC₅₀ (μM) 7-day treatment Compound UMSCC10b UMSCC10b/15s(1R,2R)-pyrodach-2 (I) 1.6 2.1 ± 0.2 (1S,2S)-pyrodach-2 (II) 1.1trans-(±)-pyrodach-2 (comparative) 5.0 (1R,2R)-pyrodach-4 (IV) 1.7 3.9

TABLE 4 IC₅₀ values for phosphaplatin compounds on human colon cancercell lines (HT-29) IC₅₀ (μM) at various IC₅₀ (μM) treatment times(CyQUANT) Compound 24 hours 7 days 72 hours (1S,2S)-pyrodach-2 (II) 10(1R,2R)-pyrodach-2 (I) 9.6 2.1 2.7 (1R,2R)-pyrodach-4 (IV) 23 cisplatin(comparative) 6.5

TABLE 5 IC₅₀ values for phosphaplatin compounds on human cancer celllines: A459, human lung adenocarcinoma cancer cells; U251, humanglioblastoma cancer cells; PC-3, human metastatic colon cancer cells;SKMEL-2, human skin melanoma cancer cells; MCF-7, human breast cancercells; OVCAR-8, human ovarian cancer cells with dysfunctional p53; andOVCAR-10, human ovarian cancer resistant to cisplatin treatment, alldetermined by CyQUANT technology, described above, that directlymeasures DNA content by measuring fluorescence signals of intercalationsIC₅₀ (μM) for various Human Cancer Cell Lines (72-hour treatment time)Compound A549 U251 PC-3 SKMEL-2 MCF-7 OVCAR-8 OVCAR-10(1R,2R)-pyrodach-2 (I) 0.9 4.6 1.7 19.7 2.3 1.2 0.8 (1S,2S)-pyrodach-2(II) 11.9 24 10.7 10 11.2 13.5 5.9 trans-(±)-pyrodach-2 11.1 10.1 24.58.3 9.4 11.8 (comparative) (1R,2R)-pyrodach-4 (IV) 6.3 3.9 20.5 34 11.417.8 (1S,2S)-pyrodach-4 (V) 21 10 19.1 4.7 15.1 35.8trans-(±)-pyrodach-4 12.2 14.6 20 14.6 12.3 23.2 (comparative) cisplatin2.8 0.75 1.08 5.34 5.1 6.3 3.1 (comparative)

Additionally these isomeric compounds were also tested by exposing themfor 144 hours in the following cell lines: UMSCC10b, Panc-1, UMSCC15s,A2780/C30 and HCC1806. The isomer (1R,2R)-pyrodach-2 exhibitedsurprisingly superior activity when compared to racemictrans-(±)-pyrodach-2. For example, (1R,2R)-pyrodach-2 exhibited an IC₅₀value of 1.7 (μM) compared to an IC₅₀ value of 18.5 (μM) for racemictrans-(±)-pyrodach-2 in pancreatic cell line Panc-1; (1R,2R)-pyrodach-2exhibited an IC₅₀ value of 0.3 (μM) compared to an IC₅₀ value of 8.9(μM) for racemic trans-(±)-pyrodach-2 in head and neck cancer cell lineUMSCC10b; and (1R,2R)-pyrodach-2 exhibited an IC₅₀ value of 9.7 (μM)compared to an IC₅₀ value of >30 (μM) for racemic trans-(±)-pyrodach-2in breast cancer cell line HCC1806.

Among the trans-isomers, clonogenic assays indicated far superioractivity of the (1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4 isomers overthe racemic mixtures trans-(±)-pyrodach-2 and trans-(±)-pyrodach-4,respectively. For example, the IC₅₀ value was found to be 180±15 μM whenthe trans-(±)-pyrodach-4 was exposed to human ovarian cancer cells(A2780) for an hour; whereas the IC₅₀ for the same cell line was foundto be 40±10 μM for (1R,2R)-pyrodach-4 under otherwise identicalexperimental conditions (Table 1). Extended exposures of (1R,2R)-isomersto a variety of human cancer cell lines yielded much lower IC₅₀ values,indicating potentials of these compounds as effective anticancer drugs.For example, (1R,2R)-pyrodach-2 has an IC₅₀ value of 500 nM (0.5 μM)from the clonogenic assay for human ovarian cell and 2 μM for theresistant human head and neck cancer.

Both enantiopure (1R,2R)-pyrodach-2 and (1S,2S)-pyrodach-2 compoundsshow equal activity within experimental error when A2780 cells wereexposed for at least 24 hr. But when cells are exposed for shorterperiod of time, e.g., 1 hour, differential activities between the(1R,2R)- and the (1S,2S)-forms were observed. The (1R,2R)-forms showsuperior activity compared to the (1S,2S)-isomer at shorter timeexposure, indicating faster uptake of the (1R,2R)-isomer by the cells.Higher IC₅₀ values for the racemic forms compared to either the (1R,2R)-or the (1S,2S)-form may indicate self-association of the two forms whichperhaps are taken by the cells at a reduced rate.

For de novo cisplatin-resistant human ovarian cancer (OVCAR-10),(1R,2R)-pyrodach-2 exhibited better in vitro efficacy than(1S,2S)-pyrodach-2. Noteworthy is that (1R,2R)-pyrodach-2 exhibitedremarkably superior activity over cisplatin in human ovarian cancer celllines OVCAR-10 (TABLES 2 and 5) and OVCAR-8 (TABLE 5).

The cis-isomer exhibited superior activity against human ovarian cancer(A2780) compared to the other compounds. For example, cis-pyrodach-2 andcis-pyrodach-4 show IC₅₀ values 300 nM and 4 μM, respectively, afterexposure to A2780 cells for 24 hours, compared with 1.0 μM and 18 μM,respectively, for the corresponding (1R,2R)-pyrodach-2 or(1R,2R)-pyrodach-4 forms. Note that cisplatin and carboplatin yieldedmuch higher IC₅₀ values, i.e., 5.0 μM and >60 μM respectively underidentical conditions.

Without intent to be bound or limited by theory, the anticipatedchemical principle teaches that the IC₅₀ value of the racemic formsshould be equal to the arithmetic average of the respective IC₅₀ valuesof the (1R,2R)-isomer and the (1S,2S)-isomer. However, as shown in thetables above, the IC₅₀ value of racemic form (for example, of pyrodach-2in A2780) is much higher than expected based on a 50/50 mixture of(1R,2R)-enantiomer and (1S,2S)-enantiomer. These data suggest thatracemic forms are less potent than expected, based on enantiomericdistribution, for unknown reasons. One plausible explanation might bethat racemic forms self-associate and, thereby, are not effectivelytaken by the cells.

In further tests, cells were treated with the platinum compoundscontinuously for 7 days. Colonies were fixed and stained using 2%crystal violet in 4% formaldehyde. Colonies containing more than 50cells were scored. The number of scored colonies from the triplicateplates was averaged, and this number was divided by the number of cellsplated to obtain a value for the fraction of cells forming colonies.These values for fraction of cells forming colonies then were correctedfor plating efficiency by dividing the fraction by the number of cellsforming colonies in plates that were not treated with platinumcompounds. The data for colony formation with A2780 are shown in FIG. 2.Note that both the (1R,2R)-pyrodach-2 and the (1S,2S)-pyrodach-2enantiomers yield almost identical IC₅₀ values of 1.0±0.1 μM for the(1R,2R) and of 1.1±0.1 μM for the (1S,2S), whereas the racemic mixtureyielded much higher IC₅₀ value of 2.4±0.2 μM indicative of lowerefficacy of the racemic mixture. Similar data for OVCAR-10 are shown inFIG. 3, comparing (1R,2R)-pyrodach-2 (RD2), (1R,2R)-pyrodach-4 (RD4),trans-(±)-pyrodach-2 (T-D2), and trans-(±)-pyrodach-4 (T-D4).

Clinical studies have shown that the (1R,2R)-enantiomer of oxaliplatin,with reference the same 1,2-diaminocylohexane carrier ligand inoxaliplatin as is present in the phosphaplatins described herein,exhibits superior efficacy over the other stereoisomers of oxaliplatin.In contrast to oxaliplatin, all three pyrophosphato isomers (i.e.,(1R,2R)-, (1S,2S)-, and cis) of both pyrodach-2 and pyrodach-4 are veryactive against a variety of cancers. There appears to be no universaltrend, however. For example, as detailed above, (1R,2R)-pyrodach-2 and(1S,2S)-pyrodach-2 showed almost equal IC₅₀ values in human A2780 cancercells when these compounds were exposed to the cells for 24 hours,whereas for de novo cisplatin-resistant human ovarian cancer (OVCAR 10),the (1R,2R)-pyrodach-2 exhibited better in vitro efficacy. On the otherhand, (1S,2S)-pyrodach-4 showed superior activity over(1R,2R)-pyrodach-4 in all human ovarian cancer cells. The cis-isomers ofpyrodach-2 and pyrodach-4 exhibited superior activity against humanovarian cancer (A2780) compared to the trans-isomers. Thus, the datareveal as a whole that some specific cancers can be treated withspecific isomers at lower doses compared to racemic forms by avoidingtoxicity from other isomers that would be present in the racemic forms.

Example 9 Monitoring of Fas Overpression by Immunofluorescence

A six-well plate with loose, pretreated cover slips was seeded withhuman ovarian cancer cells, A2780 and A2780/C30 (cross resistant to 30μM cisplatin and 100 μM carboplatin), obtained from Dr. Thomas Hamilton(Fox Chase Cancer Center, Philadelphia, Pa.) in 2.5 mL of media. Cellswere cultured as a monolayer using RPMI 1640 supplemented with 10% fetalbovine serum, 2 mM glutamine, 0.25 units/mL insulin andpenicillin/streptomycin (100 units/mL) (Fisher Scientific, Pittsburg,Pa.) in a 37° C. incubator continuously gassed with 5% CO₂.

The cells at 70% confluency were treated with one of the phosphaplatincompounds from the above Synthesis Examples for 1 hour. The plates thenwere carefully washed twice with ice-cold phosphate-buffered saline(PBS), were replaced with regular media, and were incubated for anadditional 1 hour at 37° C. with 5% CO₂. The cover slips were washedwith PBS twice, and the cells were treated with freshly prepared 1%formaldehyde and then incubated at room temperature for 5 to 7 minutes.The fixed cells were washed with PBS for three times and were blockedwith 2% FBS/PBS at 4° C. for 30 minutes, followed by washing the cellswith PBS three times for 5 minutes each wash.

Individual cover slips were removed and flipped over onto 100 μL of a1:100 dilution of FAS primary antibody (Cell Signaling Technology Inc.,Danvers, Mass.) in 5% BSA/1% milk/PBS on a Parafilm® surface for 1 hourat room temperature. Thereupon, the cover slips were transferred back toa clean six-well plate for each subsequent wash, namely three washeswith PBS, each for 5 minutes. The cover slips were flipped over a secondtime on another clean Parafilm® surface and were incubated with 100 μLof secondary FITC-antibody at a 1:500 dilution in 5% BSA/1% milk/PBS for1 hour at room temperature. The cover slips then were washed with PBSthree times at 5 minutes each. The moist cover slips then were mountedonto a microscope slide with Ultracruz® mounting media with DAPI(4′,6-diamidino-2-phenylindole) (Santa Cruz Biotechnology, Santa Cruz,Calif.) for identifying the cell nuclei. Microscopy was performed usinga fluorescence microscope at 10×, 40×, and 100× magnifications.

Example 10 Western Blot/Immunodecoration for Protein Expression

Following 12% SDS-PAGE electrophoresis, proteins were transferred to aPVDF membrane using 100 V for 1.5 hours at 4° C. Membranes were washedwith 0.05% TWEEN-20 and Tris-buffered saline (TBST) solution and blockedwith 5% non-fat dry milk and 1% BSA. The membrane was incubated with a1:25,000 dilution of primary antibody of the protein of interest overnight at 4° C. Membranes were washed with 0.05% TBST followed byincubating in the corresponding HRP-conjugated antibody (1:40,000dilution). The proteins were visualized using ECL-Advancechemiluminescent system (Amersham-GE Healthcare Biosciences, Pittsburg,Pa.).

The phosphaplatins activate a number of proapototic and tumorsuppression genes such as FAS, PTEN, PUMA, BAX and others. Experimentsthrough Western Blots confirm high levels of protein expressionstranscribed by these genes. For example, trans-(±)-pyrodach-4 treatedmice exhibit upregulation of FAS (up to 25-fold), BAX (up to 4 fold),PUMA (up to 5-fold), and down regulation of VEGFR (up to 50%) uponexposure of trans-(±)-pyrodach-4 for 1 hour to 12 hours. Likewise, BCL2was down regulated as much as 70% by RR-pyrodach-2 and RR-pyrodach-4.Additionally, FAS, FADD, and platinum compound were co-localized in thelipid rafts. These activations are also associated with the increasedexpression of Sphingomyelinase (Smase), as verified by the increasedprotein expression of SMase.

Smase hydrolyzes sphingomyelin to ceramide and phosphoryl choline. In atypical experiment, cancer cells (1×10⁶) are exposed to(1R,2R)-pyrodach-2 or (1R,2R)-pyrodach-4 (10 μM) at different timeintervals (from 5 min to 2 hr). Cells are centrifuged and cell lysate iscollected and washed with cold PBS. Amplex red reagent kit (Invitrogen)is used to monitor the Smase activity. The assays are performed based onthe recommended protocols included with the kit. Typically, 11 μL ofcell lysate is suspended in 50 mM sodium citrate buffer (pH 5.0) on a96-well plate, and sphingomyelin (5.0 mM) is added to each well. Samplesare incubated for 1 hour at 37° C. Following the incubation, 100 μL ofAmplex Red reaction solution containing 100 mM Tris-HCl (100 μM), AmplexRed (2 unit/mL) horseradish peroxidase, 0.2 unit/mL choline oxidase, and8 unit/mL alkaline phosphatase (pH 8.0), are added to each well. Samplesare then incubated for 30 minutes at 37° C. Fluorescence intensity ismeasured at 590 nm using an excitation wavelength of from 530 nm to 560nm. Fluorescence values from wells containing control samples(untreated) are subtracted from each sample measurement.

Example 11 Determination of Platinum in the Lipid Raft

Platinum content was quantitatively measured on a Graphite FurnaceAtomic Absorption Spectrometer (Perkin Elmer AA-600) from calibrationcurves established by using a platinum standard (Perkin Elmer, Waltham,Mass.) in 0.1% HNO₃. The treated phosphaplatin cells were washed with 1mL of ice-cold PBS and halt protease inhibitor, PI (Pierce, Rockford,Ill.) 4 times and the pellet was collected by centrifugation at 4° C. at1000×g between each wash. The cell pellets were brought up in 250 μL ofPBS/PI, and protein content was measured by using the micro BCA method(Pierce, Rockford, Ill.). Bovine serum albumin (BSA) prepared atdifferent concentrations was used to plot the standard curve. Quantifiedprotein samples were digested in concentrated HNO₃ for 4 hours, followedby treatment with 30% H₂O₂ for 1 hour prior to analysis.

Example 12 Toxicity Studies in SCID Mice

Female SCID mice from 4 to 5 weeks old (c.b-17/LCR-Prkdc(SCID)/Crl,Strain Code 236^(r), Charles River Labs, Wilmington, Mass.) wereacclimated for one week before initiation of toxicity trials. Viasterile 26-gauge needles and syringes, the mice were injectedintra-peritoneally (i/p) with 100 μL of one of the phosphaplatincompounds described in the above Synthesis Examples in sterile filteredPBS. The injections ranged in dosage from 5 mg/kg to 60 mg/kg andoccurred once on day 1, once on day 3, and once on day 5.

To evaluate the toxicity of the phosphaplatin compounds, adverse events(i.e., >20% weight loss and/or change in food consumption, departurefrom normal behavior, other health issues, or death) were recorded everyday. Frequency and severity of occurrence of the adverse events in themice injected with the phosphaplatin compounds was compared to the samein control groups of mice. The control groups were given a mockinjection (as a control for stress response) or an injection consistingof PBS (vehicle).

Two groups of mice were treated with commercially available platinumcompounds for comparison, with the dosage based on previously publisheddata (i.e., cisplatin at 12 mg/kg and carboplatin at 60 mg/kg) ascomparison with phosphaplatin compounds. At the end of the study, allmice were anesthetized with 358 mg/kg avertin and blood was collected bycardiac puncture using a 26-gauge needle and a tubercuilin syringe.Thereby, the mice were euthanized by exsanguination under anesthesia.After euthanasia organs including the liver, spleen, heart, lung, ovary,and kidney were harvested, stored in 10% Formalin, and paraffin blockedfor histopathological examination. Changes in tissue characteristicswere examined by a skilled pathologist.

Ovarian tumor inflicted SCID mice were treated with trans-(±)-pyrodach-2and trans-(±)-pyrodach-4 at various doses up to 40 mg/kg, and tumorgrowth was monitored up to six weeks or until the tumor size became solarge that these animals were sacrificed. The tumor growth or regressionby the phosphaplatin-treated mice was then compared withcisplatin-treated mice (7 mg/kg) and carboplatin-treated mice (60mg/kg). Three doses of platinum compounds were administered on alternatedays when the tumor grew to a size of at least 100 mm³ size. Gross andnet log cell kill values were then calculated using the formulas:

Gross  Log  cell  Kill = ((T − C))/(3.32Td)${{Net}\mspace{14mu}{Log}\mspace{14mu}{cell}\mspace{14mu}{Kill}} = \frac{\left( {T - C - {{duration}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{treatment}}} \right)}{3.32{Td}}$where T and C are median times in days to grow tumor to a specifiedsize, and Td is the median time in days to double the size of the tumorin control animals.

The gross log cell kill values recorded for three dose-regimen was foundto be greater than 3 and the log net cell kill value was greater than 2at a dose of 40 mg/kg. In contrast, cisplatin (7 mg/kg) treated mice,although showing tumor regression, died within 7 days. Carboplatintreated mice (60 mg/kg dose) displayed much lower log cell kill values(less than 2) compared to trans-(±)-pyrodach-4. Further escalation ofcarboplatin dose was not possible, because more than 50% of thepopulation had died at the 60 mg/kg dose. Based on the in vitro data, itis believed that cis-pyrodach-2, cis-pyrodach-4, (1R,2R)-pyrodach-2, and(1R,2R)-pyrodach-4 in particular would exhibit better efficacy than theracemic trans-(±)-pyrodach-4.

Example 13 In Vivo Efficacy on Ovarian Cancer Cell Line A2780

Stable clonal ovarian cancer cell lines A2780 were grown in cell cultureuntil 80-90% confluency was reached. Trypsin (GIBCO/BRL, Grand Island,N.Y.) was used to detach adherent cells. Trypsinized cells werethoroughly washed with PBS (Phosphate Buffered Saline) to removetrypsin. Efficacy of phosphaplatin compounds were evaluated using humanovarian cancer, A2780 by subcutaneous xenograft in SCID HairlessOutBred, SHO-Prkdc^(scid)Hr^(hr), (Charles River Labs, Wilmington,Mass.) mice. Female SCID mice 4 to 5 weeks old were acclimated for oneweek before initiation of efficacy trials.

Cancer cells were re-suspended in PBS, and all mice except for negativecontrol mice were injected subcutaneously with from 1×10⁶ cells/0.10 mLto 5×10⁶ cells/0.10 mL in PBS and were evaluated for tumors as afunction of time using sterile 26 gauge needles and syringes. The micewere examined daily for tumor growth. Tumors were measured using digitalcalipers. The tumor volume was calculated by the formula (W²×L)/2, whereW is the tumor measurement at the widest point, and L is the tumordimension at the longest point, where the volume of the tumor in mm³ isequivalent to the weight in mg.

After approximately 2 weeks of the subcutaneous injection of cancercells, the tumors that had reached a distinguishable tumor sizes(approximately 100-200 mm³), phosphaplatin compound administration orcontrol injections were initiated. The mice were administeredphosphaplatin compounds intra-peritoneally once on day 1, again once onday 3, and again once on day 5. Each group of xenograft mice was treatedwith phosphaplatin compounds, and a matched set was treated withvehicle/placebo (PBS solution) and mock injection (as a control forstress response). In addition, two groups of mice were treated withcommercially available platinum compounds for comparison with thephosphaplatin compounds, with dosages of the commercially availableplatinum compounds being based on previously published data (i.e.,cisplatin at 12 mg/kg and carboplatin at 60 mg/kg).

The xenograft SCID mice were monitored every day for any adverse eventsin their health by measuring weight loss/gain and food consumption.Measurements were stopped and the study was ended when the tumor sizeexceeded 3000 mm³. At the end of the study, mice were anesthetized with358 mg/kg avertin, and blood was collected by cardiac puncture using a26-gauge needle and a tubercuilin syringe. Thereby, the mice wereeuthanized by exsanguination under anesthesia. After euthanasia organsincluding the liver, spleen, heart, lung, ovary, and kidney wereharvested, stored in 10% Formalin, and paraffin blocked forhistopathological examination. Changes in tissue characteristics wereexamined by a skilled pathologist.

Efficacy data of (1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4 against humanovarian cancer A2780 in the mouse xenograft model (immunocompromised NIHIII mice) are summarized in FIG. 4. During the trial, mice were treatedevery other day for three days (“qodx3”)—once on day 1, once on day 3,and once on day 5. In particular, mice were treated with 40 mg/kg of(1R,2R)-pyrodach-2, 10 mg/kg of (1R,2R)-pyrodach-4, or of 5 mg/kgcisplatin. The numbers N in the legend of FIG. 4 report the number oftrials, over which the shown data points were derived as averages. Thedata in FIG. 4 from the first ten days of treatments with(1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4 clearly show a tumorregression of during initial stages of the treatment.

Example 14 In Vivo Efficacy on Human Ovarian Cancer and HumanHead-and-Neck Cancer

Human Ovarian cancer (OVCAR-10) is known to exhibit resistant to bothcisplatin and carboplatin. Stable human clonal ovarian cancer cell linesOVCAR-10 and head and neck cancer UMSCC10b were grown separately in cellculture until 80-90% confluency was reached. Trypsin (GIBCO/BRL, GrandIsland, N.Y.) was used to detach adherent cells. Trypsinized cells werethoroughly washed with PBS (Phosphate Buffered Saline) to removetrypsin. Efficacy of phosphaplatin compounds were evaluated bysubcutaneously implanting human OVCAR-10 and UMSCC-10b xenograft in SCIDHairless OutBred, SHO-Prkdc^(scid)Hr^(hr), (Charles River Labs,Wilmington, Mass.) mice, NIH (NIH III: NIHBNX-F; NIHS-Lystbg Foxn1nuBtkxid, 4-week old female, Taconic(Rensselaer, N.Y.) mice.

Female SCID/NIH mice 4-5 weeks old were acclimated for one week beforeinitiation of efficacy trials. Cancer cells were re-suspended in PBS andall mice except for negative control mice were injected subcutaneouslywith from 1×10⁶ cells/0.10 mL to 5×10⁶ cells/0.10 mL in PBS. The tumorsize was evaluated as a function of time. The mice were examined dailyfor tumor growth. Tumors were measured using digital calipers. The tumorvolume was calculated by the formula (W²×L)/2, where W is the tumormeasurement at the widest point, and L is the tumor dimension at thelongest point, where the volume of the tumor in mm³ is equivalent to theweight in mg.

After approximately 2 weeks of the subcutaneous injection of cancercells, the tumors that had reached a distinguishable tumor sizes(approximately 100 mm³ to 200 mm³), phosphaplatin compounds of desireddoses were administred intra-peritoneally. These administrations weredone one time on day 1, once on day 3, and once on day 5.

Each group of xenograft mice was treated with phosphaplatin compounds,and a matched set was treated with vehicle/placebo (PBS solution) andmock injection (as a control for stress response). In addition, twogroups of mice were treated with commercially available platinumcompounds for comparison with the phosphaplatin compounds, with dosagesof the commercially available platinum compounds being based onpreviously published data (i.e., cisplatin at 12 mg/kg and carboplatinat 60 mg/kg). The xenograft SCID mice/NIH were monitored every day forany adverse events in their health by measuring weight loss/gain andfood consumption. Measurements were stopped and the study was ended whenthe tumor size exceeded 3000 mm³.

At the end of the study, mice were anesthetized with 358 mg/kg avertin,and blood was collected by cardiac puncture using a 26-gauge needle anda tubercuilin syringe. The mice were euthanized by exsanguination underanesthesia. After euthanasia organs including the liver, spleen, heart,lung, ovary, and kidney were harvested, were stored in 10% Formalin, andwere paraffin blocked for histopathological examination. Changes intissue characteristics were examined by a skilled pathologist.

Efficacy data of (1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4 against humanovarian cancer cells OVCAR-10 are summarized in FIG. 5, compared with aPBS/Bicarb control. Doses were administered (40 mg/kg body weight) whenthe tumor reached 100-200 mm³ in size. The treated mice showed tumorregression during the treatment time. Measurements of tumor sizes overtime are summarized in FIG. 6 for a control (PBS/Bicarbonate)administered qodx3, carboplatin at 60 mg/kg administered qodx3, and(1R,2R)-pyrodach-4 at 40 mg/kg administered qdx3. Dotted arrowsemphasize the time (in days, interpolated as necessary) at which averagetumor size reached 1000 mm³. Compared with the control, whereascarboplatin exhibited a percent increased life-span (% ILS) of onlyabout 125%, the (1R,2R)-pyrodach-4 exhibited % ILS of about 209%.

Efficacy data of (1R,2R)-pyrodach-2 and (1R,2R)-pyrodach-4 against humanhead-and-neck cancer UMSCC10b are summarized in FIG. 7. Doses wereadministered qodx3 and/or qdx4, ranging from 10 mg/kg body weight to 40mg/kg body weight. These efficacies were compared with the racemic formtrans-(±)-pyrodach-4. The doses were administered when the tumor reached100-200 mm³ in size.

Example 15 Maximum Tolerated Doses of (1R,2R)-pyrodach-2 and(1R,2R)-pyrodach-4

To determine the maximum tolerated doses, toxicity experiments wereconducted for (1R,2R)-pyrodach-2, (1R,2R)-pyrodach-4,trans-(±)-pyrodach-4, trans-(±)-pyrodach-4, cisplatin, and carboplatin.Escalating doses were administered from 10 mg/kg to 100 mg/kg in 4-5week-old female ICR (CD-1) mice, Taconic(Rensselaer, N.Y.). The mice hadbody weights between 15 g and 24 g. The mice were injected with one ofthe phosphaplatins every other day in a three-dose regimen. Totalbody-weight loss of 20% or more, unthrifty appearance, failure to gainweight, other observable health issues, departures from normalbehaviors, or death, were considered an adverse event.

At lower doses of 10 mg/kg, no loss of weight was observed. No death orloss of greater than 20% of body weight was observed up to the highestdose of 40 mg/kg for all phosphaplatin compounds. These results can becompared to cisplatin, for which 100% of the mice died at a dose of 12mg/kg, and to carboplatin, for which 33% death was observed at a dose of60 mg/kg. At higher doses of 100 mg/kg, all mice either lost greaterthan 20% weight or died within fifteen days of administration.

Example 16 Quantitative Gene Expression from Real-Time PCR Experiments

Quantitative real-time PCR experiments were performed to estimate theexpression of a few targeted genes. Human epithelial ovarian carcercells (A2780) and cisplatin-resistant cells (A2780/C30) were cultured inRPMI 1640 with or without cisplatin, (1R,2R)-pyrodach-4, andtrans-(±)-pyrodach-4 for 0, 3, 12 and 24 hours. Both treated anduntreated cancer cells were maintained in RPMI 1640 medium with 10%fetal bovine serum, 2 μmol L-glutamine, 100 units/mLpenicillin-streptomycin and 0.25 units/mL insulin solution, at 37° C.with 5% CO₂. RNA was isolated from cells, both the treated anduntreated. Treated cells are those cells harvested after the exposure ofcells to a phosphaplatin at its IC₅₀ value concentration at differenttime intervals, from three hours to 24 hrs.

The RNA samples were treated with DNase-free RNase (QIAGEN Sciences) toremove DNA. Then, cDNA was synthesized using the High CapacityRNA-to-cDNA kit (Applied Biosystems) according to the manufacturer'sinstructions. The quantity and purity of RNA were determined by UVspectroscopy (NanoDrop 2000 Thermo Scientific, Wilmington, Del.). Aminimum absorbance ratio index (ratio of the absorbance measured at 260nm over the absorbance measured at 280 nm) of 1.9 was used as anacceptable purity.

The isolated RNA samples were stored at −80° C. and were subjected tominimal freeze-thaw cycles to maintain RNA integrity. Duplex real-timePCRs were performed using Taqman® Gene Expression Assays for the targetgene and endogenous control (β-actin) in the same reaction well. Theendogenous control is the reference used to normalize the target mRNA.These chain reactions were initially performed using different cDNAconcentrations to determine the optimal concentrations of cDNA requiredto detect the gene of interest. The target gene was labeled with a bluedye (FAM, absorbance: 494 nm, emission: 518 nm) while the reference genewas tagged with a green dye (VIC, absorbance: 538 nm, emission: 554 nm).The cDNA concentrations were selected such that the threshold cycle (Ct)values for the target genes were between 17 and 32, most sensitive forthe fluorescence detection by ABI StepOnePlus™ instrument used for themeasurements.

The threshold cycle number (Ct), the point at which the fluorescence ofthe qPCR reaction just exceeds the threshold fluorescence of thedetection system, was determined. These Ct values were used to comparethe levels of target genes and the endogenous controls. Quantitativegene expressions are reported in terms of fold-change (2^(−ΔΔCt)) whichwere calculated from ΔCt and ΔΔCt values according to the relationships:ΔCt=(Ct_(target)−Ct_(reference)) andΔΔCt=(ΔCt)_(time X)−(ΔCt)_(time zero (control)). The fold-expression forthe control samples (untreated) remained equal to 1, because the valueof ΔΔCt=0, and, therefore, 2⁰=1.

This application should not be considered limited to the specificexamples and embodiments described herein, but rather should beunderstood to cover all aspects of the invention. Various modifications,equivalent processes, as well as numerous structures and devices towhich the present invention may be applicable will be readily apparentto those of skill in the art. Those skilled in the art will understandthat various changes may be made without departing from the scope of theinvention, which is not to be considered limited to what is described inthe specification.

What is claimed is:
 1. A phosphaplatin complex selected from the groupconsisting of: (iii) cis-pyrodach-2 having formula (III),

or pharmaceutically acceptable salt or solvate thereof; and (vi)cis-pyrodach-4 having formula (VI),

or pharmaceutically acceptable salt or solvate thereof.
 2. A compositionfor treating a proliferative disease, said composition comprising: (a)one or more phosphaplatin complexes selected from the group consistingof: (iii) cis-pyrodach-2 having formula (III):

or pharmaceutically acceptable salts or solvates thereof; (vi)cis-pyrodach-4 having formula (VI):

or pharmaceutically acceptable salts or solvates thereof; and (vii) amulticomplex mixture of said phosphaplatin complexes of formula (III)and (VI) and (b) at least one pharmaceutically acceptable ingredientselected from the group consisting of carriers, diluents, adjuvants, andvehicles.
 3. The composition of claim 2, wherein said phosphaplatincomplexes are isolated, monomeric phosphaplatin complexes.
 4. Thecomposition of claim 2, further comprising enantioenriched pyrodach-2having an enantiomeric excess of from 10% to 100% of (1R,2R)-pyrodach-2.5. The composition of claim 4, wherein said enantioenriched pyrodach-2consists of (1R,2R)-pyrodach-2 having formula (I) and (1S,2S)-pyrodach-2having formula (II):


6. The composition of claim 2, further comprising enantioenrichedpyrodach-2 having an enantiomeric excess of from 10% to 100% of(1S,2S)-pyrodach-2.
 7. The composition of claim 6, wherein saidenantioenriched pyrodach-2 consists of (1R,2R)-pyrodach-2 having formula(I) and (1S,2S)-pyrodach-2 having formula (II):


8. The composition of claim 2, further comprising enantioenrichedpyrodach-4 having an enantiomeric excess of from 10% to 100% of(1R,2R)-pyrodach-4.
 9. The composition of claim 8, wherein saidenantioenriched pyrodach-4 consists of (1R,2R)-pyrodach-4 having formula(IV) and (1S,2S)-pyrodach-4 having formula (V):


10. The composition of claim 2, further comprising enantioenrichedpyrodach-4 having an enantiomeric excess of from 10% to 100% of(1S,2S)-pyrodach-4.
 11. The composition of claim 10, wherein saidenantioenriched pyrodach-4 consists of (1R,2R)-pyrodach-4 having formula(IV) and (1S,2S)-pyrodach-4 having formula (V):


12. The composition of claim 2, comprising cis-pyrodach-2.
 13. Thecomposition of claim 2, comprising cis-pyrodach-4.
 14. The compositionof claim 2, wherein said proliferative disease is a cancer.
 15. Thecomposition of claim 2, wherein said proliferative disease is selectedfrom ovarian cancer, testicular cancer, small-cell lung cancer,non-small-cell lung cancer, head-and-neck cancers, skin cancer,pancreatic cancer, breast cancer, glioblastoma cancer, and colon cancer.16. The composition of claim 2, wherein said proliferative disease is acancer resistant to treatment by at least one of cisplatin, carboplatin,and oxaliplatin.